Penicillin G acylase (benzylpenicillin amidohydrolase, also named penicillin amidase; EC 3.5.1.11) is an enzyme used commercially to hydrolyse penicillin G or 3-desacetoxycephalosporin G to phenylacetic acid and 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7-ADCA), respectively, the most important intermediates for the industrial production of semi-synthetic penicillins and cephalosporins. This enzyme also catalyses the reverse reaction, viz. the N-acylation of 6-APA and 7-ADCA with organic esters to generate their corresponding N-acetylated penicillin and 3-cephem compounds, respectively. See the reviews of Vandamme, E.J., In: Microbial Enzymes and Bioconversions, E.H. Rose (Ed.), Economic Microbiology 5, 467-552 (1980); and P.B. Mahajan, Appl. Biochem. Biotechnol. 1, 83-86 (1982).
Various types of microorganisms have been proposed in the literature as Penicillin G acylase producing strains useful for the deacylation of penicillin G and 3-desacetoxycephalosporin G. Examples of such acylase producing microorganisms are certain strains of the species Escherichia coli, Kluyvera citrophila and Proteus rettgeri. It is to be noted that some penicillin G acylase activity has been described in the whole cell fraction of Alcaligenes faecalis (C.A. Claveridge et al., Nature 4733, 237-238 (1960)). However, no enzyme or some enzymes responsible for this activity from A faecalis have been described up to now.
The use of recombinant DNA methods has enabled an increase of the production levels of commercially used penicillin G acylases (Mayer et al., Adv. Biotechnol. 1, 83-86 (1982)) and has enlarged the insight into the processing of these enzymes (Schumacher et al., Nucleic Acids Res. 14, 5713-5727 (1986)). The penicillin G acylase of E. coli was found to be produced as a large precursor protein, which was further processed into the periplasmic mature protein constituting a small (.alpha.) and a large (.beta.) subunit. Cloning and sequencing of the Kluyvera citrophila acylase gene has revealed a close harmony with the E. coli acylase gene (J.L. Barbero et al., Gene 49, 69-80 (1986)). Also for the Proteus rettgeri penicillin G acylase gene a small and a large subunit has been described (G.O. Daumy et al., Gene 49, 69-80 (1986); Spanish patent application No. 8602933).